Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.

H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide-substrate RNA pairs.

H/ACA Small Ribonucleoproteins: Structural and Functional Comparison Between Archaea and Eukaryotes.

During ribosome synthesis, ribosomal RNA is modified through the formation of many pseudouridines and methylations which contribute to ribosome function across all domains of life. In archaea and eukaryotes, pseudouridylation of rRNA is catalyzed by H/ACA small ribonucleoproteins (sRNPs) utilizing different H/ACA guide RNAs to identify target uridines for modification. H/ACA sRNPs are conserved in archaea and eukaryotes, as they share a common general architecture and function, but there are also several notable differences between archaeal and eukaryotic H/ACA sRNPs. Due to the higher protein stability in archaea, we have more information on the structure of archaeal H/ACA sRNPs compared to eukaryotic counterparts. However, based on the long history of yeast genetic and other cellular studies, the biological role of H/ACA sRNPs during ribosome biogenesis is better understood in eukaryotes than archaea. Therefore, this review provides an overview of the current knowledge on H/ACA sRNPs from archaea, in particular their structure and function, and relates it to our understanding of the roles of eukaryotic H/ACA sRNP during eukaryotic ribosome synthesis and beyond. Based on this comparison of our current insights into archaeal and eukaryotic H/ACA sRNPs, we discuss what role archaeal H/ACA sRNPs may play in the formation of ribosomes.

Assaying the Molecular Determinants and Kinetics of RNA Pseudouridylation by H/ACA snoRNPs and Stand-Alone Pseudouridine Synthases.

Posttranscriptional modifications of RNA play an important role in promoting the maturation and functional diversity of many RNA species. Accordingly, understanding the enzymes and mechanisms that underlie RNA modifications is an important aspect in advancing our knowledge of the continually expanding RNA modification field. However, of the more than 160 currently identified RNA modifications, a large portion remains without quantitative detection assays for their biochemical characterization. Here, we describe the tritium release assay as a convenient tool allowing for the quantitative assessment of in vitro RNA pseudouridylation by stand-alone or box H/ACA RNA-guided pseudouridine synthases. This assay enables quantification of RNA pseudouridylation over a time course to effectively compare pseudouridylation activity between different substrates and/or different recombinant enzymes as well as to determine kinetic parameters. With the help of a quench-flow apparatus, the tritium release assay can be adapted for rapid kinetic measurements under single-turnover conditions to dissect reaction mechanisms. As examples, we show the formation of pseudouridines by a reconstituted Saccharomyces cerevisiae H/ACA small ribonucleoprotein (snoRNP) and an Escherichia coli stand-alone pseudouridine synthase.